Selectivity, mainly governed by column chemistry, is the key in HPLC separation. Although reversed-phase columns (e.g., C18) are most commonly used in pharmaceutical applications, they often fail to retain highly polar molecules (e.g., counter ions), and offer limited selectivities. Ion exchange (IEX) chromatography is used to separate ionic or ionizable molecules. However, it has limited use in small-molecule separations due to inadequate hydrophobic retention. Ion pairing chromatography can help to overcome the aforementioned difficulties, but it often requires extended equilibration time, a complicated mobile phase that is incompatible with MS, and a dedicated column.
Mixed-mode chromatography provides a viable solution to these challenges by using both reversed phase and ion-exchange retention mechanisms. One major advantage of this approach is that column selectivity can easily be modified by adjusting mobile phase ionic strength, pH and/or organic solvent concentration. As the result, not only is the selectivity of a mixed-mode column complementary to that of reversed-phase columns, but it also allows for the development of multiple complementary selectivities on a given column under different appropriate conditions. Mixed-mode chromatography is well-suited to retaining ionic analytes, hydrophobic (e.g., Naproxen) or hydrophilic (e.g., Na+ and Cl− ions), and requires no ion-pairing agents in the method, significantly improving MS compatibility. This technique has been growing rapidly because of its advantages over conventional chromatography, such as its high resolution, adjustable selectivity, high sample loading, and no need for ion-pairing agents. Many applications involving hydrophilic ionizable compounds that are problematic on a C18 column are easily addressed on a mixed-mode column.
Mixed-mode media can be classified into four general categories. Recently, mixed-modal chromatography which is based on at least two modes of interaction, in most cases ion-exchange and hydrophobic interaction, have become more popular, because it seems that often the achieved resolution outperforms that of corresponding separate individual single-mode chromatographic separations. Such mixed-modal chromatography can be carried out in a number of different variants, which have been reviewed by L. W. McLaughlin (1989) in Chem. Rev. 89, pages 309-319:
Category 1 utilized in-line coupling of columns packed with different individual single mode separation materials. For example, a reversed-phase column can be coupled in-line to an ion-exchange column.
Category 2 is based on the use of mixed-bed columns. The columns include a blend of distinct separation materials, such as ion-exchange and reversed-phase particles in a single HPLC column. Blending two types of different materials such as RP particles and anion-exchanger (e.g., strong anion-exchanger particles) in a single column has been used as a method to combine different retention mechanisms, and such columns are commercially available.
Category 3 materials include distinct interactive functionalities such as an ion-exchange moiety and hydrophobic moiety on different components of the separation material. For example, one separation moiety can be located on the dedicated chromatographic ligand and the other on the support. The different interactive moieties are spatially separated.
Category 4 mixed-modal chromatographic materials have the two (or more) distinct interaction sites on a single chromatographic ligand.
Exemplary mixed mode media are based on bonded silica modified by a mixture of both RP and IEX ligands in the bonding step. Although these materials seem straightforward to synthesize, their use in many applications is limited by selectivity drifting. This drift is due mainly to the difference in hydrolytic stability between the RP and IEX ligand bonded sites. Newer mixed-mode media use functional silyl ligands that contain both RP and IEX functionalities to covalently modify silica particles. While the constant ratio between RP and IEX bonded sites greatly improves, a pronounced difference in selectivity exists between the two interaction sites.
Compositions useful in chromatographic separations, as well as methods of making and using these compositions, have been created and are described herein.